Compositions and methods for diagnosing and treating an inflammation

ABSTRACT

An isolated polypeptide is provided. The polypeptide comprising an antigen recognition domain capable of specifically binding a human scavenger receptor, wherein the antigen recognition domain comprises at least three CDR amino acid sequences selected from the group consisting of SEQ ID NO: 11, 15, 19, 23, 27 and 31. Also provided are compositions which comprise the peptide and uses of same.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.11/991,711 filed on Mar. 7, 2008, which is a National Phase of PCTPatent Application No. PCT/IL2006/001059 filed on Sep. 11, 2006, whichis a continuation in part (CIP) of U.S. patent application Ser. No.11/222,745 filed on Sep. 12, 2005, now U.S. Pat. No. 8,017,113. Thecontents of the above applications are incorporated herein by reference.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 56550SequenceListing.txt, created on Jun. 9,2013, comprising 11,661 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to antibodies, compositions and methodsfor diagnosing and treating inflammation. More particularly, the presentinvention relates to the use of anti scavenger receptor antibodies intreatment of an inflammatory response.

Inflammation is a physiological condition characterized in the acuteform by the classical signs of pain, heat, redness, swelling and loss offunction. Inflammation often accompanies diseases such as MultipleSclerosis (MS), osteoarthritis, Inflammatory Bowl Disease (IBD)including Crohn's disease and ulcerative colitis, Rheumatoid Arthritis(RA), SLE, type I diabetes (IDDM), atherosclerosis, encephalomyelitis,Alzheimer's disease, stroke, traumatic brain injury, Parkinson'sdisease, septic shock and others. In most cases, there is no effectivecure for inflammation associated with such disease and existingtreatments are palliative and largely fail to control the underlyingcauses of tissue degradation.

Scavenger receptors (SRs) are cell surface proteins, which are typicallyfound on macrophages and bind various types of chemically modifiedlipoproteins (1-3), such as low-density lipoprotein (LDL). This familyof trans-membrane receptors which are highly varied in structure areinvolved in receptor-mediated endocytosis, phagocytosis of apoptoticcells and bacteria, as well as in cell adhesion [Peiser L. et al., Curr.Opin. Immun. 14(1):123-128, 2002]. Since the massive receptor-mediateduptake of cholesterol from modified LDL can convert cultured macrophagesinto cholesteryl ester-filled foam cells, similar to those found inatherosclerotic plaques, it has been postulated that these receptorsalso function in deposition of LDL cholesterol of macrophages in arterywalls during the initial stages of atherosclerotic plaque formation [1].

Scavenger receptors (SRs) are termed as such since they mediate thebinding of remarkably wide variety of polyanionic ligands [e.g.,modified proteins, sulfated polysaccharides and certain polynucleotides[1, 3, 4]. This property led to the hypothesis that these receptors forma part of an in innate immune response by serving as pattern recognitionreceptors that bind a wide variety of pathogen components [2-5].

SR-B 1 (also referred to as SR-BI or CLA-I) is a macrophage scavengermolecule and a receptor for high-density lipoprotein (HDL) [2, 3, 6, 7]that mediates cholesterol uptake from cells [Rigotti A. et al., Curr.Opin. Lipidol., 8:181-8, 1997; Rigotti A. et al., Proc. Natl. Acad.Sci., 94:12610-5, 1997]. SR-B1 can also serve as a receptor for non-HDLlipoproteins and appears to play an important role in reversecholesterol transport. In vivo experiments showed that this receptor isimportant for HDL metabolism in mice, and for the metabolism of LDL andHDL cholesterol in humans [Stang H. et al., J. Biol. Chem. 274:32692-8.,1999; Swarnakar S. et al., J. Biol. Chem. 274:29733-9., 1999]. Studiesinvolving the manipulation of SR-B1 gene expression in mice, indicatethat its expression protects against atherosclerosis [Kozarsky K. F.,and Krieger M., Curr. Opin. Lipidol. 10:491-7., 1999; Ueda Y. et al., J.Biol. Chem. 275:20368-73., 2000; Acton S. L. et al., Mol. Med. Today5:518-24., 1999]. It was also suggested that HDL and particularly itsprotein fraction Apo-A1 affect the in vitro production ofpro-inflammatory mediators by macrophages (8). Among mediators derivedfrom macrophages that propagate inflammation are interleukin 12 (IL-12),TNF-α and possibly IL-6 whereas, TGF-β and IL-10 have predominantlyanti-inflammatory effects [Kiefer R. et al., Prog. Neurobiol.64(2):109-27, 2001].

PCT Publication No. WO 2004/041179 teaches targeting of scavengerreceptor SR-B1 (Cla-I) for the treatment of infectious diseasesassociated with invasion of foreign antigens such as bacterial or viralantigens (e.g., infection, sepsis and associated inflammation). This isbased on the discovery that peptides with an amphipathic helical motifblock cellular uptake of LPS (lipopolysaccharide) and proinflammatoryresponses induced by LPS, LTA (lipoteichoic acid) and bacterial cpn60(Chaperonin 60) and amyloid peptides in vitro. Thus the inventors of PCTPublication No. WO 2004/041179 conclude that agents with an amphipathicmotif targeting SR-BI (scavenger receptor class B type I) couldpotentially be used to treat sepsis, bacterial and viral infections andinflammatory diseases where LPS, LTA, viral envelope proteins, and/orheat shock proteins contribute to pathogenesis.

WO 2004/041179 does not suggest the above-described agents for thefavourable treatment of autoimmune diseases (which are not associatedwith foreign pathogenic agents such as LPS, nor with amyloid) such asIBD. Nor does the art teach the use of SR-B 1 specific antibodysequences having an anti-inflammatory activity activity for thetreatment of inflammatory diseases in general and autoimmune diseases inparticular, such as multiple sclerosis.

There is thus, a widely recognized need for and it would be highlyadvantageous to have novel agents and methods using same for targetingSR-B 1 and treating autoimmune diseases.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anisolated polypeptide comprising an antigen recognition domain capable ofspecifically binding a human scavenger receptor, wherein the antigenrecognition domain comprises at least three CDR amino acid sequencesselected from the group consisting of SEQ ID NO: 11, 15, 19, 23, 27 and31.

According to another aspect of the present invention there is providedan isolated polypeptide comprising an antigen recognition domain capableof specifically binding a human scavenger receptor, wherein the antigenrecognition domain comprises CDR amino acid sequences as set forth inSEQ ID NO: NO: 11, 15, 19, 23, 27 and 31.

According to yet another aspect of the present invention there isprovided an isolated polynucleotide comprising a nucleic acid sequenceencoding the polypeptide.

According to still another aspect of the present invention there isprovided a pharmaceutical composition comprising as an active ingredientthe polypeptide.

According to an additional aspect of the present invention there isprovided a method of reducing inflammation in a subject in need thereof,the method comprising administering to the subject a therapeuticallyeffective amount of the polypeptide, thereby reducing the inflammationin the subject.

According to yet an additional aspect of the present invention there isprovided use of the polypeptide for the manufacture of a medicamentidentified for treating IBD.

According to still an additional aspect of the present invention thereis provided use of the polypeptide for the manufacture of a medicamentidentified for treating multiple sclerosis.

According to a further aspect of the present invention there is provideduse of the polypeptide for the manufacture of a medicament identifiedfor treating an autoimmune disease.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing novel compositions andmethods containing same for diagnosing and treating an inflammatoryresponse.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a photograph depicting cross-reactivity of monoclonal antiSR-B1 antibody, E12, with human and mouse orthologs. Recombinantproteins were resolved on SDS-PAGE and transferred to nitrocellulosemembrane. The membrane was subjected to E12;

FIG. 2 is a graph depicting dose-dependent induction of IL-10 secretionfrom cultured peritoneal macrophages treated with E12;

FIG. 3 is a bar graph depicting dose-dependent suppression of NO levelsin cultured peritoneal macrophages treated with E12;

FIG. 4 is a graph depicting the effect of E12 (closed squares), controlisotype matching antibody (circles) or no treatment on ongoing EAE inmice induced with such, as determined by reduction in EAE score;

FIGS. 5 a-c are bar graphs depicting the effect of E12 (pink) or controlantibodies (grey) on cytokine secretion from spleen cells of 19 dayEAE-induced mice. FIG. 5 a—IL-4. FIG. 5 b—IL-12. FIG. 5 c—IL-10;

FIGS. 6 a-f are photographs showing IL-10 immunostaining of Lumbarspinal cord sections from EAE induced mice (19 days of disease onset)subjected to no treatment (FIG. 6 a), or treated with E12 (FIG. 6 b), orisotype matching control antibody (FIG. 6 c). FIGS. 6 a-c shows stainingwith biotinylated E12 for presence of scavenger receptor expressingcells. FIGS. 6 d-f shows staining with anti IL-10 antibody. Anti-SR-BItherapy reduces the histological score of EAE;

FIGS. 7 a-e are photographs showing representative histological colonsections obtained at day 12 of disease onset from naïve rats (FIG. 7 a),positive control rats suffering form TNBS induced IBD (FIG. 7 b), ratssuffering from TNBS induced IBD that were subjected to repeatedadministration of isotype matched control IgG (FIG. 7 c) in comparisonto those treated with mAb E12 (FIGS. 7 d-e); and

FIGS. 8 a-i show representative immuno-histological sections obtained atday 12 of disease onset from control rats suffering from TNBS inducedIBD (FIGS. 8 a-c), rats suffering form TNBS induced IBD that weresubjected to repeated administration of isotype matched control IgG(FIGS. 8 d-f) in comparison to diseased rats treated with mAb E12 (FIGS.8 g-i). FIGS. 8 a, d and g are stained with mAb ED 1 (macrophagesbio-marker); FIGS. 8 b, e and h are stained with anti CD3 (T cellbio-marker) and FIGS. 8 c, f and I are stained with an anti IL-10 mAb.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of compositions and methods which can be usedfor the treatment of inflammation. Specifically, the present inventionrelates to the use of anti scavenger receptor antibodies in treatinginflammatory response.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Diseases and disorders which have significant inflammatory componentsare ubiquitous. Skin disorders, bowel disorders, certain degenerativeneurological disorders, arthritis, autoimmune diseases and otherillnesses afflict many patients. The factors underlying these disordersare varied and include infectious agents, autoimmune factors, dietary orenvironmental factors and genetic factors. In the majority of cases, thecausative elements have not been defined and many of the keypathophysiological components have not been elucidated. Accordingly,treatment options for the majority of these diseases is suboptimal.

The present inventor has previously shown that the immune system canselectively generate autoimmunity to chemokines and otherproinflammatory mediators when such a response is beneficial for thehost [9, 10, 11, 12, 14, 15]. For example, patients suffering fromrheumatoid arthritis (RA) but not osteoarthritis (OA) have significantlevels of autoantibodies directed to TNF-α, and therapies thatneutralize the function of TNF-α suppress RA but not OA. Studiesconducted by the present inventor have shown that selectiveamplification of these beneficial antibodies by targeted DNA vaccinesprovided protective immunity in experimental models (9, 10, 11, 12, 14,15). The present inventor have further shown that subjects sufferingfrom inflammatory disease exhibit elevated levels of autoantibodies toscavenger receptor (SR) and showed that DNA vaccination against SR-B 1can prevent such diseases by altering the cytokine profile produced bymacrophages from pro-inflammatory cytokines to anti-inflammatorycytokines (see WO2004/080385).

It is now reported that the present inventor also developed, throughlaborious experimentations and screening a novel therapeutic anti-SR-B1monoclonal antibody, E12, which is capable of altering the cytokineprofile and inflammatory activities of macrophages. This antibody whichwas sequenced is directed against a surface exposed epitope of thescavenger receptor (FIG. 1) and is cross-reactive to human CLA-I (humanSR-B1) and also affects the cytokine profile and in vitro activities ofhuman macrophages (a cell line) and as such can be used as a valuabletherapeutic and diagnostic tool (see Example 1 and FIGS. 1-3). Thisantibody was also shown effective in suppressing ongoing EAE and TNBSinduced IBD (see FIGS. 4-8). Immunohistological analysis clearly showedthat in both diseases anti SR-BI therapy altered the cytokine productionof invading leukocytes, at the autoimmune site, into high IL-10producing cells. This may explain significant therapeutic effect of thisantibody in these diseases. Immunohistological analysis of CNS sectionsusing anti SR-BI mAb also showed that SR-BI positive leukocytes enterthe site of inflammation (so far detected only for EAE). Thus, it issuggested that anti SR-BI antibodies described herein affect thecytokine profile and inflammatory functions of inflammatory leukocytesentering the autoimmune site, and thereby the function and polarizationof autoimmune T cells there.

These findings suggest that the present antibody can be used fortargeting scavenger receptor and for treatment of inflammatory diseases,especially IBD and multiple sclerosis.

Thus according to one aspect of the present invention there is providedan isolated polypeptide comprising an antigen recognition domain capableof specifically binding a human scavenger receptor, wherein said antigenrecognition domain comprises at least three CDR amino acid sequencesselected at least 90% homologous to the group consisting of SEQ ID NOs.:11, 15, 19, 23, 27 and 31.

According to one embodiment of this aspect of the present invention thepolypeptide comprises the CDR amino acid sequences are as set forth inSEQ ID NOs.: 11, 15, 19, 23, 27 and 31.

Preferably, the polypeptide is an antibody. More preferably the antibodyis capable of eliciting an anti-inflammatory activity. As used herein“an anti-inflammatory activity” refers to any reduction in immune cellinflammatory activity, such as reduction in pro-inflammatory cytokine(such as TNF-a, IL-1 and IL-12) secretion preferably accompanied byinduction of anti-inflammatory cytokine (e.g., IL-10, IL-4 and TGF-b)secretion.

The term “antibody” refers to whole antibody molecules as well asfunctional fragments thereof, such as Fab, F(ab′)₂, and Fv that arecapable of binding with antigenic portions of the target polypeptide.These functional antibody fragments constitute preferred embodiments ofthe present invention, and are defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule that can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule as described in, for example,U.S. Pat. No. 4,946,778.

Methods of generating antibodies are well known in the art. Purificationof serum immunoglobulin antibodies (polyclonal antisera) or reactiveportions thereof can be accomplished by a variety of methods known tothose of skill including, precipitation by ammonium sulfate or sodiumsulfate followed by dialysis against saline, ion exchangechromatography, affinity or immunoaffinity chromatography as well as gelfiltration, zone electrophoresis, etc. (see Goding in, MonoclonalAntibodies: Principles and Practice, 2nd ed., pp. 104-126, 1986,Orlando, Fla., Academic Press). Under normal physiological conditionsantibodies are found in plasma and other body fluids and in the membraneof certain cells and are produced by lymphocytes of the type denoted Bcells or their functional equivalent. Antibodies of the IgG class aremade up of four polypeptide chains linked together by disulfide bonds.The four chains of intact IgG molecules are two identical heavy chainsreferred to as H-chains and two identical light chains referred to asL-chains. Additional classes include IgD, IgE, IgA, IgM and relatedproteins.

Methods of generating and isolating monoclonal antibodies are well knownin the art, as summarized for example in reviews such as Tramontano andSchloeder, Methods in Enzymology 178, 551-568, 1989. A recombinantscavenger receptor polypeptide may be used to generate antibodies invitro (see Example 6 of the Examples section which follows). In general,a suitable host animal is immunized with the recombinant polypeptide.Advantageously, the animal host used is a mouse of an inbred strain.Animals are typically immunized with a mixture comprising a solution ofthe recombinant polypeptide in a physiologically acceptable vehicle, andany suitable adjuvant, which achieves an enhanced immune response to theimmunogen. By way of example, the primary immunization conveniently maybe accomplished with a mixture of a solution of the recombinantpolypeptide and Freund's complete adjuvant, said mixture being preparedin the form of a water in oil emulsion. Typically the immunization willbe administered to the animals intramuscularly, intradermally,subcutaneously, intraperitoneally, into the footpads, or by anyappropriate route of administration. The immunization schedule of theimmunogen may be adapted as required, but customarily involves severalsubsequent or secondary immunizations using a milder adjuvant such asFreund's incomplete adjuvant. Antibody titers and specificity of bindingto the polypeptide can be determined during the immunization schedule byany convenient method including by way of example radioimmunoassay, orenzyme linked immunosorbant assay, which is known as the ELISA assay.When suitable antibody titers are achieved, antibody-producinglymphocytes from the immunized animals are obtained, and these arecultured, selected and cloned, as is known in the art. Typically,lymphocytes may be obtained in large numbers from the spleens ofimmunized animals, but they may also be retrieved from the circulation,the lymph nodes or other lymphoid organs. Lymphocytes are then fusedwith any suitable myeloma cell line, to yield hybridomas, as is wellknown in the art. Alternatively, lymphocytes may also be stimulated togrow in culture, and may be immortalized by methods known in the artincluding the exposure of these lymphocytes to a virus, a chemical or anucleic acid such as an oncogene, according to established protocols.After fusion, the hybridomas are cultured under suitable cultureconditions, for example in multi-well plates, and the culturesupernatants are screened to identify cultures containing antibodiesthat recognize the hapten of choice. Hybridomas that secrete antibodiesthat recognize the recombinant polypeptide are cloned by limitingdilution and expanded, under appropriate culture conditions. Monoclonalantibodies are purified and characterized in terms of immunoglobulintype and binding affinity.

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment.

Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly. These methods are described, for example,by Goldenberg, in U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety (see also Porter, R. R., Biochem. J., 73: 119-126, 1959).Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of V_(H) and V_(L) chains. Thisassociation may be noncovalent, as described in Inbar et al. (Proc.Nat'l Acad. Sci. USA 69:2659-62, 1972). Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise V_(H) and V_(L) chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the V_(H) and V_(L)domains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426,1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al.,U.S. Pat. No. 4,946,778, all of which are hereby incorporated byreference in its entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick and FryMethods, 2: 106-10, 1991).

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues form a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues, which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin [Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art(see also Example 6 of the Examples section). Generally, a humanizedantibody has one or more amino acid residues introduced into it from asource, which is non-human. These non-human amino acid residues areoften referred to as import residues, which are typically taken from animport variable domain. Humanization can be essentially performedfollowing the method of Winter and co-workers [Jones et al., Nature,321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodentCDRs or CDR sequences for the corresponding sequences of a humanantibody. Accordingly, such humanized antibodies are chimeric antibodies(U.S. Pat. No. 4,816,567), wherein substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites in rodentantibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human monoclonal antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

Antibodies of the present invention can be encoded from isolatedpolynucleotides which comprise nucleic acid sequences such as set forthin SEQ ID NOs. 12, 16, 20, 24, 28 and 32.

Polypeptides of the present invention can be synthesized using solidphase peptide synthesis procedures which are well known in the art andfurther described by John Morrow Stewart and Janis Dillaha Young, SolidPhase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).Synthetic peptides can be purified by preparative high performanceliquid chromatography [Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.] and the composition ofwhich can be confirmed via amino acid sequencing.

In cases where large amounts of the polypeptides are desired, they canbe generated using recombinant techniques such as described by Bitter etal., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990)Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514,Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J.3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al.(1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988,Methods for Plant Molecular Biology, Academic Press, NY, Section VIII,pp 421-463.

As mentioned, the polypeptides (also referred to herein as agents) ofthe present invention may be used for reducing or treating aninflammatory response (i.e., inflammation) in a subject.

As used herein the term “treating” refers to preventing, curing,reversing, attenuating, alleviating, minimizing, suppressing or haltingthe deleterious effects of an inflammatory response.

As used herein the phrase “inflammatory response” refers to an immuneresponse which results in inflammation, typically occurring as a resultof injurious stimuli including infection, burns, trauma, neoplasia,autoimmune signals and exposure to chemicals, heat or cold or any otherharmful stimulus. An inflammatory response according to the presentinvention refers to an acute phase response and a chronic inflammation.

As used herein the term “subject” refers to subject who may benefit fromthe present invention such as a mammal (e.g., canine, feline, ovine,porcine, equine, bovine, human), preferably a human subject.

The method of this aspect of the present invention is effected byproviding to a subject in need thereof a therapeutically effectiveamount of the polypeptide of the present invention, thereby reducing theinflammatory response in the subject.

As used herein a “scavenger receptor” refers to a gene product (i.e.,RNA or protein) of a scavenger receptor, which is known in the Art.Examples of scavenger receptors include but are not limited to class Ascavenger receptors, class B scavenger receptors and class F scavengerreceptors. The scavenger receptor is preferably one which is expressedand displayed by macrophages. Preferably, the scavenger receptor of thepresent invention is SR-BI, a member of the CD36 family, GenBankAccession No. NP_(—)005496, also known as CLA-I or SR-B1.

Scavenger receptor activity refers to cell adhesion activity,transporter activity, apoptotic activity, lipid metabolism activity,signal transduction activity and/or preferably cytokine secretionactivity.

An effector of a scavenger receptor refers to an endogenous moleculewhich up-regulates or activates scavenger receptor activity. Forexample, an effector can be a modified lipid (e.g., oxidized lipid,glycated lipid, alkylated lipid, nitrated lipid, acetylated lipid),which binds to the scavenger receptor and activates signaling therefrom.

The above-described agents can be provided to the subject per se, or aspart of a pharmaceutical composition where they are mixed with apharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the preparationaccountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases. One of the ingredients included in thepharmaceutically acceptable carrier can be for example polyethyleneglycol (PEG), a biocompatible polymer with a wide range of solubility inboth organic and aqueous media (Mutter et al. (1979).

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,inrtaperitoneal, intranasal, or intraocular injections. Alternately, onemay administer a preparation in a local rather than systemic manner, forexample, via injection of the preparation directly into a specificregion of a patient's body.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, for oralingestion by a patient. Pharmacological preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carbomethylcellulose; and/or physiologically acceptable polymerssuch as polyvinylpyrrolidone (PVP). If desired, disintegrating agentsmay be added, such as cross-linked polyvinyl pyrrolidone, agar, oralginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use. The preparation of the present invention mayalso be formulated in rectal compositions such as suppositories orretention enemas, using, e.g., conventional suppository bases such ascocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro assays. For example, a dose can be formulated in animal modelsand such information can be used to more accurately determine usefuldoses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions including the preparation of the present inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition.

Pharmaceutical compositions of the present invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert.

A number of diseases and conditions, which typically cause inflammatoryresponse in individuals can be treated using the methodology describedhereinabove.

Examples of such diseases and conditions are summarized infra.

Inflammatory Diseases

Include, but are not limited to, chronic inflammatory diseases and acuteinflammatory diseases.

Inflammatory Diseases Associated with Hypersensitivity

Examples of hypersensitivity include, but are not limited to, Type Ihypersensitivity, Type II hypersensitivity, Type III hypersensitivity,Type IV hypersensitivity, immediate hypersensitivity, antibody mediatedhypersensitivity, immune complex mediated hypersensitivity, T lymphocytemediated hypersensitivity and DTH.

Type I or immediate hypersensitivity, such as asthma.

Type II hypersensitivity include, but are not limited to, rheumatoiddiseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V.et al., Histol Histopathol 2000 July; 15 (3):791), spondylitis,ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3):189), systemic diseases, systemic autoimmune diseases, systemic lupuserythematosus (Erikson J. et al., Immunol Res 1998; 17 (1-2):49),sclerosis, systemic sclerosis (Renaudineau Y. et al., Clin Diagn LabImmunol. 1999 March; 6 (2):156); Chan O T. et al., Immunol Rev 1999June; 169:107), glandular diseases, glandular autoimmune diseases,pancreatic autoimmune diseases, diabetes, Type I diabetes (Zimmet P.Diabetes Res Clin Pract 1996 October; 34 Suppl:S125), thyroid diseases,autoimmune thyroid diseases, Graves' disease (Orgiazzi J. EndocrinolMetab Clin North Am 2000 June; 29 (2):339), thyroiditis, spontaneousautoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec.15; 165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al., NipponRinsho 1999 August; 57 (8):1810), myxedema, idiopathic myxedema (MitsumaT. Nippon Rinsho. 1999 August; 57 (8):1759); autoimmune reproductivediseases, ovarian diseases, ovarian autoimmunity (Garza K M. et al., JReprod Immunol 1998 February; 37 (2):87), autoimmune anti-sperminfertility (Diekman A B. et al., Am J Reprod Immunol. 2000 March; 43(3):134), repeated fetal loss (Tincani A. et al., Lupus 1998; 7 Suppl2:S107-9), neurodegenerative diseases, neurological diseases,neurological autoimmune diseases, multiple sclerosis (Cross A H. et al.,J Neuroimmunol 2001 Jan. 1; 112 (1-2):1), Alzheimer's disease (Oron L.et al., J Neural Transm Suppl. 1997; 49:77), myasthenia gravis (InfanteA J. And Kraig E, Int Rev Immunol 1999; 18 (1-2):83), motor neuropathies(Kornberg A J. J Clin Neurosci. May; 7 (3):191), Guillain-Barresyndrome, neuropathies and autoimmune neuropathies (Kusunoki S. Am JMed. Sci. 2000 April; 319 (4):234), myasthenic diseases, Lambert-Eatonmyasthenic syndrome (Takamori M. Am J Med. Sci. 2000 April; 319(4):204), paraneoplastic neurological diseases, cerebellar atrophy,paraneoplastic cerebellar atrophy, non-paraneoplastic stiff mansyndrome, cerebellar atrophies, progressive cerebellar atrophies,encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis,Sydeham chorea, Gilles de la Tourette syndrome, polyendocrinopathies,autoimmune polyendocrinopathies (Antoine J C. and Honnorat J. Rev Neurol(Paris) 2000 January; 156 (1):23); neuropathies, dysimmune neuropathies(Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl1999; 50:419); neuromyotonia, acquired neuromyotonia, arthrogryposismultiplex congenita (Vincent A. et al., Ann N Y Acad. Sci. 1998 May 13;841:482), cardiovascular diseases, cardiovascular autoimmune diseases,atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl 2:S135),myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2:S132),thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9),granulomatosis, Wegener's granulomatosis, arteritis, Takayasu'sarteritis and Kawasaki syndrome (Praprotnik S. et al., Wien KlinWochenschr 2000 Aug. 25; 112 (15-16):660); anti-factor VIII autoimmunedisease (Lacroix-Desmazes S. et al., Semin Thromb Hemost. 2000; 26(2):157); vasculitises, necrotizing small vessel vasculitises,microscopic polyangiitis, Churg and Strauss syndrome,glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis,crescentic glomerulonephritis (Noel L H. Ann Med Interne (Paris). May;151 (3):178); antiphospholipid syndrome (Flamholz R. et al., J ClinApheresis 1999; 14 (4):171); heart failure, agonist-like β-adrenoceptorantibodies in heart failure (Wallukat G. et al., Am J Cardiol. 1999 Jun.17; 83 (12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med.Int. 1999 April-June; 14 (2):114); hemolytic anemia, autoimmunehemolytic anemia (Efremov D G. et al., Leuk Lymphoma 1998 January; 28(3-4):285), gastrointestinal diseases, autoimmune diseases of thegastrointestinal tract, intestinal diseases, chronic inflammatoryintestinal disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000January; 23 (1):16), celiac disease (Landau Y E. and Shoenfeld Y.Harefuah 2000 Jan. 16; 138 (2):122), autoimmune diseases of themusculature, myositis, autoimmune myositis, Sjogren's syndrome (Feist E.et al., Int Arch Allergy Immunol 2000 September; 123 (1):92); smoothmuscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999June; 53 (5-6):234), hepatic diseases, hepatic autoimmune diseases,autoimmune hepatitis (Manns M P. J Hepatol 2000 August; 33 (2):326) andprimary biliary cirrhosis (Strassburg C P. et al., Eur J GastroenterolHepatol. 1999 June; 11 (6):595).

Type IV or T cell mediated hypersensitivity, include, but are notlimited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevittH O. Proc Natl Acad Sci USA 1994 Jan. 18; 91 (2):437), systemicdiseases, systemic autoimmune diseases, systemic lupus erythematosus(Datta S K., Lupus 1998; 7 (9):591), glandular diseases, glandularautoimmune diseases, pancreatic diseases, pancreatic autoimmunediseases, Type 1 diabetes (Castano L. and Eisenbarth G S. Ann. Rev.Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves'disease (Sakata S. et al., Mol Cell Endocrinol 1993 March; 92 (1):77);ovarian diseases (Garza K M. et al., J Reprod Immunol 1998 February; 37(2):87), prostatitis, autoimmune prostatitis (Alexander R B. et al.,Urology 1997 December; 50 (6):893), polyglandular syndrome, autoimmunepolyglandular syndrome, Type I autoimmune polyglandular syndrome (HaraT. et al., Blood. 1991 Mar. 1; 77 (5):1127), neurological diseases,autoimmune neurological diseases, multiple sclerosis, neuritis, opticneuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May;57 (5):544), myasthenia gravis (Oshima M. et al., Eur J Immunol 1990December; 20 (12):2563), stiff-man syndrome (Hiemstra H S. et al., ProcNatl Acad Sci USA 2001 Mar. 27; 98 (7):3988), cardiovascular diseases,cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al., J ClinInvest 1996 Oct. 15; 98 (8):1709), autoimmune thrombocytopenic purpura(Semple J W. et al., Blood 1996 May 15; 87 (10):4245), anti-helper Tlymphocyte autoimmunity (Caporossi A P. et al., Viral Immunol 1998; 11(1):9), hemolytic anemia (Sallah S. et al., Ann Hematol 1997 March; 74(3):139), hepatic diseases, hepatic autoimmune diseases, hepatitis,chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol1990 March; 54 (3):382), biliary cirrhosis, primary biliary cirrhosis(Jones D E. Clin Sci (Colch) 1996 November; 91 (5):551), nephricdiseases, nephric autoimmune diseases, nephritis, interstitial nephritis(Kelly C J. J Am Soc Nephrol 1990 August; 1 (2):140), connective tissuediseases, ear diseases, autoimmune connective tissue diseases,autoimmune ear disease (Yoo T J. et al., Cell Immunol 1994 August; 157(1):249), disease of the inner ear (Gloddek B. et al., Ann N Y Acad Sci1997 Dec. 29; 830:266), skin diseases, cutaneous diseases, dermaldiseases, bullous skin diseases, pemphigus vulgaris, bullous pemphigoidand pemphigus foliaceus.

Examples of delayed type hypersensitivity include, but are not limitedto, contact dermatitis and drug eruption.

Examples of types of T lymphocyte mediating hypersensitivity include,but are not limited to, helper T lymphocytes and cytotoxic Tlymphocytes.

Examples of helper T lymphocyte-mediated hypersensitivity include, butare not limited to, T_(h)1 lymphocyte mediated hypersensitivity andT_(h)2 lymphocyte mediated hypersensitivity.

Autoimmune Diseases

Include, but are not limited to, cardiovascular diseases, rheumatoiddiseases, glandular diseases, gastrointestinal diseases, cutaneousdiseases, hepatic diseases, neurological diseases, muscular diseases,nephric diseases, diseases related to reproduction, connective tissuediseases and systemic diseases.

Examples of autoimmune cardiovascular diseases include, but are notlimited to atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl2:S135), myocardial infarction (Vaarala 0. Lupus. 1998; 7 Suppl 2:S132),thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9), Wegener'sgranulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S.et al., Wien Klin Wochenschr 2000 Aug. 25; 112 (15-16):660), anti-factorVIII autoimmune disease (Lacroix-Desmazes S. et al., Semin ThrombHemost. 2000; 26 (2):157), necrotizing small vessel vasculitis,microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focalnecrotizing and crescentic glomerulonephritis (Noel L H. Ann Med Interne(Paris). 2000 May; 151 (3):178), antiphospholipid syndrome (Flamholz R.et al., J Clin Apheresis 1999; 14 (4):171), antibody-induced heartfailure (Wallukat G. et al., Am J. Cardiol. 1999 Jun. 17; 83 (12A):75H),thrombocytopenic purpura (Moccia F. Ann Ital Med. Int. 1999 April-June;14 (2):114; Semple J W. et al., Blood 1996 May 15; 87 (10):4245),autoimmune hemolytic anemia (Efremov D G. et al., Leuk Lymphoma 1998January; 28 (3-4):285; Sallah S. et al., Ann Hematol 1997 March; 74(3):139), cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al.,J Clin Invest 1996 Oct. 15; 98 (8):1709) and anti-helper T lymphocyteautoimmunity (Caporossi A P. et al., Viral Immunol 1998; 11 (1):9).

Examples of autoimmune rheumatoid diseases include, but are not limitedto rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 July;15 (3):791; Tisch R, McDevitt H O. Proc Natl Acad Sci units S A 1994Jan. 18; 91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al.,Arthritis Res 2001; 3 (3): 189).

Examples of autoimmune glandular diseases include, but are not limitedto, pancreatic disease, Type I diabetes, thyroid disease, Graves'disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto'sthyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmuneanti-sperm infertility, autoimmune prostatitis and Type I autoimmunepolyglandular syndrome. diseases include, but are not limited toautoimmune diseases of the pancreas, Type 1 diabetes (Castano L. andEisenbarth G S. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res ClinPract 1996 October; 34 Suppl:S125), autoimmune thyroid diseases, Graves'disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 June; 29(2):339; Sakata S. et al., Mol Cell Endocrinol 1993 March; 92 (1):77),spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol2000 Dec. 15; 165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al.,Nippon Rinsho 1999 August; 57 (8):1810), idiopathic myxedema (Mitsuma T.Nippon Rinsho. 1999 August; 57 (8):1759), ovarian autoimmunity (Garza KM. et al., J Reprod Immunol 1998 February; 37 (2):87), autoimmuneanti-sperm infertility (Diekman A B. et al., Am J Reprod Immunol. 2000March; 43 (3):134), autoimmune prostatitis (Alexander R B. et al.,Urology 1997 December; 50 (6):893) and Type I autoimmune polyglandularsyndrome (Hara T. et al., Blood. 1991 Mar. 1; 77 (5):1127).

Examples of autoimmune gastrointestinal diseases include, but are notlimited to, chronic inflammatory intestinal diseases (Garcia Herola A.et al., Gastroenterol Hepatol. 2000 January; 23 (1):16), celiac disease(Landau Y E. and Shoenfeld Y. Harefuah 2000 Jan. 16; 138 (2):122),colitis, ileitis and Crohn's disease.

Examples of autoimmune cutaneous diseases include, but are not limitedto, autoimmune bullous skin diseases, such as, but are not limited to,pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

Examples of autoimmune hepatic diseases include, but are not limited to,hepatitis, autoimmune chronic active hepatitis (Franco A. et al., ClinImmunol Immunopathol 1990 March; 54 (3):382), primary biliary cirrhosis(Jones D E. Clin Sci (Colch) 1996 November; 91 (5):551; Strassburg C P.et al., Eur J Gastroenterol Hepatol. 1999 June; 11 (6):595) andautoimmune hepatitis (Manns M P. J Hepatol 2000 August; 33 (2):326).

Examples of autoimmune neurological diseases include, but are notlimited to, multiple sclerosis (Cross A H. et al., J Neuroimmunol 2001Jan. 1; 112 (1-2):1), Alzheimer's disease (Oron L. et al., J NeuralTransm Suppl. 1997; 49:77), myasthenia gravis (Infante A J. And Kraig E,Int Rev Immunol 1999; 18 (1-2):83; Oshima M. et al., Eur J Immunol 1990December; 20 (12):2563), neuropathies, motor neuropathies (Kornberg A J.J Clin Neurosci. 2000 May; 7 (3):191); Guillain-Barre syndrome andautoimmune neuropathies (Kusunoki S. Am J Med. Sci. 2000 April; 319(4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. AmJ Med. Sci. 2000 April; 319 (4):204); paraneoplastic neurologicaldiseases, cerebellar atrophy, paraneoplastic cerebellar atrophy andstiff-man syndrome (Hiemstra H S. et al., Proc Natl Acad Sci units S A2001 Mar. 27; 98 (7):3988); non-paraneoplastic stiff man syndrome,progressive cerebellar atrophies, encephalitis, Rasmussen'sencephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles dela Tourette syndrome and autoimmune polyendocrinopathies (Antoine J C.and Honnorat J. Rev Neurol (Paris) 2000 January; 156 (1):23); dysimmuneneuropathies (Nobile-Orazio E. et al., Electroencephalogr ClinNeurophysiol Suppl 1999; 50:419); acquired neuromyotonia, arthrogryposismultiplex congenita (Vincent A. et al., Ann N Y Acad. Sci. 1998 May 13;841:482), neuritis, optic neuritis (Soderstrom M. et al., J NeurolNeurosurg Psychiatry 1994 May; 57 (5):544) and neurodegenerativediseases.

Examples of autoimmune muscular diseases include, but are not limitedto, myositis, autoimmune myositis and primary Sjogren's syndrome (FeistE. et al., Int Arch Allergy Immunol 2000 September; 123 (1):92) andsmooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother1999 June; 53 (5-6):234).

Examples of autoimmune nephric diseases include, but are not limited to,nephritis and autoimmune interstitial nephritis (Kelly C J. J Am SocNephrol 1990 August; 1 (2):140).

Examples of autoimmune diseases related to reproduction include, but arenot limited to, repeated fetal loss (Tincani A. et al., Lupus 1998; 7Suppl 2:S107-9).

Examples of autoimmune connective tissue diseases include, but are notlimited to, ear diseases, autoimmune ear diseases (Yoo T J. et al., CellImmunol 1994 August; 157 (1):249) and autoimmune diseases of the innerear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec. 29; 830:266).

Examples of autoimmune systemic diseases include, but are not limitedto, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998;17 (1-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin DiagnLab Immunol. 1999 March; 6 (2):156); Chan O T. et al., Immunol Rev 1999June; 169:107).

Infectious Diseases

Examples of infectious diseases include, but are not limited to, chronicinfectious diseases, subacute infectious diseases, acute infectiousdiseases, viral diseases, bacterial diseases, protozoan diseases,parasitic diseases, fungal diseases, mycoplasma diseases and priondiseases.

Graft Rejection Diseases

Examples of diseases associated with transplantation of a graft include,but are not limited to, graft rejection, chronic graft rejection,subacute graft rejection, hyperacute graft rejection, acute graftrejection and graft versus host disease.

Allergic Diseases

Examples of allergic diseases include, but are not limited to, asthma,hives, urticaria, pollen allergy, dust mite allergy, venom allergy,cosmetics allergy, latex allergy, chemical allergy, drug allergy, insectbite allergy, animal dander allergy, stinging plant allergy, poison ivyallergy and food allergy.

Cancerous Diseases

Examples of cancer include but are not limited to carcinoma, lymphoma,blastoma, sarcoma, and leukemia. Particular examples of cancerousdiseases but are not limited to: Myeloid leukemia such as Chronicmyelogenous leukemia. Acute myelogenous leukemia with maturation. Acutepromyelocytic leukemia, Acute nonlymphocytic leukemia with increasedbasophils, Acute monocytic leukemia. Acute myelomonocytic leukemia witheosinophilia; Malignant lymphoma, such as Birkitt's Non-Hodgkin's;Lymphoctyic leukemia, such as Acute lumphoblastic leukemia. Chroniclymphocytic leukemia; Myeloproliferative diseases, such as Solid tumorsBenign Meningioma, Mixed tumors of salivary gland, Colonic adenomas;Adenocarcinomas, such as Small cell lung cancer, Kidney, Uterus,Prostate, Bladder, Ovary, Colon, Sarcomas, Liposarcoma, myxoid, Synovialsarcoma, Rhabdomyosarcoma (alveolar), Extraskeletel myxoidchonodrosarcoma, Ewing's tumor; other include Testicular and ovariandysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignantmelanoma, Mesothelioma, breast, skin, prostate, and ovarian.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Therapeutic Monoclonal Human Anti SR-B1 Antibody

A monoclonal human anti SR-B 1 antibody was produced for therapeuticuse.

Materials and Methods

SR-B1 Encoding Plasmids:

DNA encoding human SR-B 1 (CLA-I) was amplified using sense primer: 5′CCATGGGCTGCTCCGCCAAA 3′ (SEQ ID NO: 6), and anti-sense primer: 5′CTACAGTTTTGCTTCCTGCAG 3′ (SEQ ID NO: 7) The above described reactionmixture was subjected to an amplification program of 1 min at 95° C., 1min at 55° C. and 1 min at 72° C. for 25 cycles, generating 1.53 kb DNAfragment of SEQ ID NO:8 (Homo sapiens encoding SR-B1 mRNA, nucleotides70-1599 from accession number:Z22555). After PCR reaction, the mixturewas loaded onto a 5% polyacrylamide gel in TAE buffer. PCR product wasgel-purified, cloned into a pUC57/T vector (T-cloning kit K1212; MBIFermentas, Vilnius, Lithuania) and then used to transform E. coli cells.Clones were then sequenced (Sequenase version 2; Upstate Biotechnology,Cleveland, Ohio) and transferred into a pcDNA3 vector (Invitrogen, SanDiego, Calif.). Large-scale preparation of plasmid DNA was conductedusing Mega prep (Qiagen, Chatsworth, Calif.).

Cells:

HEK293 (ATCC) were transfected with human SR-B 1 as described before[Scarselli E, et al., EMBO J. 21(19):5017-25, 2002]. Expression wasverified by FACS analysis as described before [Scarselli E, et al., EMBOJ. 21(19):5017-25, 2002].

Production of Monoclonal Human Anti SR-B1 Antibody:

Human anti SR-B 1 monoclonal antibodies were produced according to oneof the two following protocols:

Protocol I

C57/B6 mice were subsequently immunized (3 weekly immunizations) withthe human SR-B1 (SEQ ID NO:8) encoding DNA plasmid. Two weeks after thelast administration, these mice were subjected to active induction ofEAE. Spleen cells were obtained for production of monoclonal antibodiestwo weeks later with SP2 cells (ATCC) as a fusion partner as describedbefore (E. Harlow & D. Lane, Antibodies, Cold Spring Harbor LaboratoryPress, 1998). Screening of positive hybridoma was done in two steps ofselection. The first one selected positive antibodies producing cellsaccording to the ability to bind the recombinant SR-B 1 over expressedby HEK293. Supernatant isolated from hybridoma clones (1000 wells) wasthen subjected to FACS analysis for their ability to bind SR-B1

Protocol II

The cloned human SR-B 1 (SEQ ID NO:8), obtained as described above, wasre-cloned into a pQE expression vector, expressed in E. coli (Qiagen)and then purified by an NI-NTA-supper flow affinity purification of6×His proteins (Qiagen). After purification, the purity of therecombinant human SR-B 1 was verified by gel electrophoresis followed bysequencing (N terminus) by the Technion's sequencing services unit(Technion, Haifa, Israel). This recombinant human SR-B 1 was theninjected into 10-weeks old BALB/C mice. First immunization was of 50 μgpeptide emulsified in CFA [incomplete Freund's adjuvant (IFA)supplemented with 10 mg/ml heat-killed Mycobacterium tuberculosis H37Rain oil; Difco Laboratories, Detroit, Mich.] at a total volume of 400 μlinto the peritoneal cavity. Later on, in a 3 weeks interval these micewere administrated with 50 μg/400 μl or recombinant human SR-B1emulsified in IFA (Difco Laboratories, Detroit, Mich.). Three weeksafter the third interval mice were injected (intravenous) with 50 μg ofrecombinant human SR-B 1 in 100 μl PBS. Three days later spleen cellswere obtained and preparation of monoclonal antibodies was conducted asdescribed above.

ELISA

The indirect ELISA was used to screen hybridomas for antibodies againstSR-BI, as follows. Ninety six-well microtiter plates (NUNC) were coatedwith 50 ng/ml of immuno (recombinant) SR-BI (SEQ ID NO: 8) in phosphatebuffered saline (PBS) overnight at 4° C., followed by blocking with 200μl of 5% BSA in PBS. Then 100 μl of hybridoma supernatants were addedand incubated for 1 hr at room temperature (RT). The plates were washed4 times with PBS containing 0.05% Tween 20 (PBS-T), and thensupplemented with peroxidase-conjugated goat anti-mouse IgG antibody for1 hr at RT, and washed 5 times with PBS-T. Then 100 μl of substratesolution 3,3′,5,5′-tetramethyl Benzedrine liquid (ICN biomedical INC,Germany, TMB) were added. The reaction was stopped using 2.5M H₂SO₄ andthe absorbance was read by an ELISA reader at a wavelength of 450 nm andbackground of 630 nm.

Cell Binding Assay

HEK 293 cell line was stably transfected with pcDNA encoding SR-BI(pcSR-BI). Positive clones were selected using neomycin (G418). Thepositively isolated ELISA hybridoma clones (isolated as described above)were taken into the second screen. Ninety six well disposable flexiblepolyvinyl chloride microtitration plates (Dynatech laboratories,Virginia) were seeded with 1*10⁶ pcSR-BI-expressing HEK 293 cells. Thecells were washed twice with PBS before 100 μl of hybridomassupernatants were added for 30 minutes on ice. Following 3 washes withPBS, peroxidase-conjugated goat anti-mouse IgG antibody was added foradditional 20 min on ice. Following 3 washes with PBS 100 μl ofsubstrate solution (TMB) was added. The reaction was stopped using 2.5MH₂SO₄. After a short centrifugation, the reaction was transferred into aclean well and the absorbance was read by an ELISA reader at awavelength of 450 nm and background of 630 nm.

Example 2 In Vitro Characterization of Anti SR-BI Therapeutic Antibodies

Human SR-B 1 cross-reactive antibodies (with CLA-I) generated asdescribed in Example 1 according to protocol 2 were in-vitro screenedand characterized. The most successful antibody obtained was E12, whichwas further in-vitro characterized as further described hereinbelow.

Materials and Experimental Procedures

Immunoblot Analysis

For single-label immunohistochemistry, standard methodology was usedwhereby sections were incubated with primary antibodies (1:100),followed by incubation with secondary antibodies (1:100). Mouse IgG andrabbit polyclonal IgG were used as control antibodies

Isotype Analysis

Isotype analysis was done using Serotec kit www.serotec.com.

Culture of Peritoneal Macrophages

Resident macrophages were obtained from a peritoneal lavage with PBS.Elicited macrophages were harvested 5 days following i.p. injection of 3ml of 3% Thioglycollate (TG, Difco, Livonia, Mich.). Peritoneal exudatecells were washed, re-suspended in RPMI 1640 medium supplemented with10% FCS, 1% penicillin, 1% streptomycin, and incubated in 24 flat-bottomplates (10⁶ cells per well in 1 ml) for overnight at 37° C. Nonadherentcells were then removed by vigorous washing (three times), andmacrophages monolayers were incubated for 1-10 days in antibiotic-freeRPMI containing 10% FCS. Fresh medium was provided every 3 days.

IL-10 Production by Macrophage Culture

The peritoneal macrophages generated as described above were treatedwith mAb E12 with or without 0.5 μg/ml LPS (Sigma) for 24 hr at 37° C.Supernatants from either treated or untreated macrophages were assayedfor the presence of IL-10 or using immunoenzimatic ELISA kits(Biolegend).

Nitrite Production by Macrophage Culture

Nitrite formation measurement was done according as described by[Katakura, T., M. Miyazaki, M. Kobayashi, D. N. Herndon, and F. Suzuki.(2004). CCL17 and IL-10 as effectors that enable alternatively activatedmacrophages to inhibit the generation of classically activatedmacrophages. J Immunol 172:1407]. Peritoneal macrophages (1*10⁶/ml) wereseeded in 24-well plates as describe above. Following treatment with LPSand/or mAb E12 the supernatant was taken and NO production was assayedby measuring the accumulation of nitrite in the culture medium by Griessreaction using Griess reagent system kit (Promega). Briefly: an equalvolume of Griess reagent (Sulfanilamide Solution) and macrophagesupernatants was incubated for 10 min at RT in a dark room. An equalvolume of N-1-napthylethylenediamine dihydrochloride (NED) was thenadded for 10 min. An ELISA reader measured the absorbance at 550 nm.Nitrite concentration was determined using NaNO₂ as a standard.

Results

Isotype analysis of E12 revealed it to be IgG1. The purified E12 wasreacted with a nitrocellulose membrane containing various recombinantproteins. As shown in FIG. 1, mAb E12 cross reacted with SR-BI and CLA-1but not with MIP, CXCL6 or IL-27. These results indicate that theantibody specifically recognizes scavenger B1 receptor in across-species dependent manner and is capable of recognizing thedenatured form of the protein indicating that it is directed against anexposed epitope of the native protein, as further demonstrated by itsability to neutralize SR-B 1 activity.

The ability of E12 to elicit anti-inflammatory activity, was in vitroassayed on cultured peritoneal macrophages. As shown in FIG. 2 culturedperitoneal macrophages treated for 24 hours with 0.5 μg/ml LPS and withmAb E12, or with isotype matched control IgG, produced significantlyhigher IL-10 in the presence of increasing amounts of E12 than comparedto control treated cells.

These results were substantiated when following NO levels in thepresence of E12 antibody and LPS (0.5 μg/ml). As shown in FIG. 3, mAbE12 suppressed NO synthesis (as determined by nitrite levels) byperitoneal macrophages in a dose dependent manner. Control matchedisotypes had no effect of NO levels.

The variable regions of E12 heavy chain (VH) and light chain (VK) weresequenced and their CDR composition determined. SEQ ID NO: 9 and 10 showthe amino acid and nucleic acid sequences of framework 1 (FWR1) of E12light chain, respectively. SEQ ID NO: 11 and 12 show the amino acid andnucleic acid sequences of CDR1 of E12 light chain, respectively. SEQ IDNO: 13 and 14 show the amino acid and nucleic acid sequences offramework 2 (FWR2) of E12 light chain, respectively. SEQ ID NO: 15 and16 show the amino acid and nucleic acid sequences of CDR2 of E12 lightchain, respectively. SEQ ID NO: 17 and 18 show the amino acid andnucleic acid sequences of framework 3 (FWR3) of E12 light chain,respectively. SEQ ID NO: 19 and 20 show the amino acid and nucleic acidsequences of CDR3 of E12 light chain, respectively.

SEQ ID NO: 21 and 22 show the amino acid and nucleic acid sequences offramework 1 (FWR1) of E12 heavy chain, respectively. SEQ ID NO: 23 and24 show the amino acid and nucleic acid sequences of CDR1 of E12 heavychain, respectively. SEQ ID NO: 25 and 26 show the amino acid andnucleic acid sequences of framework 2 (FWR2) of E12 heavy chain,respectively. SEQ ID NO: 27 and 28 show the amino acid and nucleic acidsequences of CDR2 of E12 heavy chain, respectively. SEQ ID NO: 29 and 30show the amino acid and nucleic acid sequences of framework 3 (FWR3) ofE12 heavy chain, respectively. SEQ ID NO: 31 and 32 show the amino acidand nucleic acid sequences of CDR3 of E12 heavy chain, respectively.

Example 3 A Monoclonal Antibody to SR-B1 is Capable of SuppressingOngoing EAE and IBD

The monoclonal antibody generated as taught in Example 1 above was shownhighly effective in suppressing ongoing EAE and TNBS induced IBD, asfurther described hereinbelow.

Materials and Methods

Induction of EAE in Mice and Suppression of the Ongoing Disease with mAbto SR-B1

A group of 18 C57BL/6 mice was subjected to MOGp35-55 induced EAE. Atthe onset of disease (day 13) these mice were separated into threeequally sick groups. On this day and on days 15 and 17 these groups wereintraveneously administered with 500 μg E12 mAb, isotype matched humanIgG (IgG1), or PBS and followed for clinical manifestation of disease(FIG. 4) by an observer blind to the experimental protocol.

Spinal Cord Histopathology

Histological examination of H&E-stained sections of formalin-fixed,paraffin-embedded sections of the lower thoracic and lumbar regions ofthe spinal cord was performed. Each section was evaluated withoutknowledge of the treatment status of the animal. The following scale wasused: 0, no mononuclear cell infiltration; 1, one to five perivascularlesions per section with minimal parenchymal infiltration; 2, five to 10perivascular lesions per section with parenchymal infiltration; and3, >10 perivascular lesions per section with extensive parenchymalinfiltration. The mean histological score±SE was calculated for eachtreatment group

Immunohistochemistry

For single-label immunohistochemistry, standard methodology was usedwhereby sections were incubated with primary antibodies (1:100),followed by incubation with secondary antibodies (1:100). Mouse IgG andrabbit polyclonal IgG were used as control antibodies

Induction of experimental Colitis in Lewis Rats

Experimental colitis was induced by intrarectal instillation of 250 μlof 125 mg/ml 2,4,6-trinitrobenzene sulfonic acid (TNBS) solution (Fluka,cat#92822) dissolved in 50% ethanol, using 8 cm neonate feeding tube asdescribed before [Fiorucci, S. et al., Immunity, 17:769., 2002]. 24hours post injection all rats developed bloody diarrhea and severediarrhea in the next day, accompanied with continuous loss of weight.

Treatment Protocol for Antibody Transfer

On days 6, 8 and 10 post induction of experimental colitis, 500 μg ofmAb E12 was injected intravenously via a tail vein. Human IgG1 (Sigma)was used as a control antibody.

Sample Collection

On day 12, the rats were killed under ketamine-xylasine anesthesia. Theterminal colon was then stripped, gently washed with PBS, openedlongitudinally and macroscopically evaluated according to a modificationof the criteria described by Morris Gut (2004); 53; 99-107. Colonicinjury was scored on a 0 (normal colon) to 5 (severe damage) scale, (seeTable 2, below).

Colon Histopathology

Tissues (terminal colon, mesentery lymph nodes and spleens) were fixedin 10% neutral buffered formalin and embedded in paraffin. Hematoxylinand eosin stained sections of the colon were evaluated histologicallyfor four parameters: extent of ulceration, submucosal infiltration,crypt abscesses and wall thickening (see Table 3). The sum of all scoresdetermined a rating of slight to severe colonic inflammation.

Immunohistochemistry

Serial sections from formalin-fixed, paraffin-embedded specimens weredeparaffinized and rehydrated in decreasing concentrations of ethylalcohol. Tissue sections were incubated with fresh 3% H₂O₂ in methanolfor 10 min and then washed with PBS. Sections were then treated bymicrowave for 15 min in 90° C. in citrate buffer and blocked with 10%donkey serum for 30 min. Immunoistochemical analysis was carried outusing primary antibodies against rat IL-10 (polyclonal goat anti ratIL-10, R&D), CD3 (mAb mouse anti rat, Pharmingen) and ED1 (mAb mouseanti rat, Serotec) over night at 4° C. in a humidified chamber.Biotinylated donkey anti goat or anti mouse IgG were used as secondaryantibodies, followed by a streptavidin-horseradish peroxidase (Zymed).The reaction was developed using aminoethylcarbazole substrate kit(Zymed).

TABLE 2 Macroscopic assessment of colonic damage Macroscopic damageScore No damage 0 Hyperemia but no ulcers 1 Fibrosis but no ulcers 2Ulceration/necrosis <1 cm 3 Ulceration/necrosis <2 cm 4Ulceration/necrosis >2 cm 5

TABLE 3 Microscopic assessment of colonic inflammation Histologicalappearance Score Extent of ulceration No ulcer 0 Small ulcers (<3 mm)1-2 Large ulcers (>3 mm) 3-5 Submucosal infiltration None 0 Mild 1Moderate 2-3 Severe 4-5 Crypt abscesses None 0 Rare 1-2 Diffuse 3-5 Wallthickness (μm) <470 0 <600 1 <700 2 <800 3 <900 4 >900 5

Results

Anti SR-BI mAb Suppress Long-Term Ongoing EAE

Three groups of mice models of EAE displaying similar clinicalmanifestations were subjected to monoclonal antibody therapy and controltreatments. As shown in FIG. 4, mice treated with PBS or control IgGcontinued to develop severe EAE, while those treated with the anti SR-BImAb E12 went into fast remission without residual sign of disease (FIG.4).

On day 19, spleen cells were isolated from representative mice of eachgroup and cultured for 72 h with the target antigen with which diseasewas induced. Levels of IL-10, IL-12 (p40 subunit) and IL-4 were thenrecorded using commercially available ELISA kits. FIGS. 5 a-c summarizethe results of this experiment showing a marked elevation in IL-10production (p<0.001), a significant elevation in IL-4 production(p<0.01) accompanied by a significant reduction in IL-12 production(p<0.01). These results are consistent with the in vitro properties ofthis antibody (see FIGS. 5 a-c and may explain, at least in part thebeneficial effect of this therapy (FIG. 4).

Spinal cord (lumbar spinal cord) sections obtained on day 19 fromcontrol EAE mice and from those subjected to IgG1 or E12 therapy (seeFIG. 4) were subjected to an immunohistological analysis of theexpression of SR-BI on leukocytes around high endothelial venules (HEV).FIGS. 6 a-c show representative sections of untreated control EAE mice,EAE mice treated with E12 and EAE mice treated with control IgG1,respectively. In all sections of sick mice leukocytes entering the CNShighly expressed SR-BI. The reduction in the density of these cells inanti SR-BI treated mice could be explained, in part, by the reducednumber of invading leukocytes resulting from the decrease in theinflammatory process (i.e. lower histological score).

Finally representative sections from these groups were subjected toimmunohistological analysis of IL-10, using a commercially availableanti IL-10 mAb. FIGS. 6 d-f show representative sections of untreatedcontrol EAE mice, EAE mice treated with E12 and EAE mice treated withcontrol IgG1, respectively. The elevation in IL-10 production insections of mice treated with E12 is apparent compared to each of thecontrol groups. These results support the above in-vitro results,substantiating the anti-inflammatory role of anti SR-B1 therapy.

Anti SR-BI mAb Suppresses Experimental Colitis

Similar analysis of the effect of anti-SR-B 1 monoclonal antibodies onIBD was effected on a rat model of colotis. The following summarizesmacroscopic and microscopic analyses on colitis induced rats (6 rats pergroup), as well as representative samples of histopathological analysis,followed by immunohistochemistry detection of ED1 positive cells(macrophages), CD3⁺ T cells and IL-10 staining in all groups.

Table 4 below clearly shows that a significant reduction in macroscopicand microscopic scores of disease which is accompanied by a markedreduction in histopathological changes in the colon.

TABLE 4 TNBS control TNBS TNBS IgG E12mAb Mean macroscopic assessment  4± 0.66  4.2 ± 0.66 2.66 ± 0.5* Mean microscopic assessment 15 ± 2 17.5 ±2.2  6.5 ± 2** *p < 0.01, **p < 0.001

FIGS. 7 a-e show representative histological colon sections obtained atday 12 of IBD onset from naïve rats (FIG. 7 a), positive control ratssuffering form TNBS induced IBD (FIG. 7 b), rats suffering from TNBSinduced IBD that were subjected to repeated administration of isotypematched control IgG (FIG. 7 c) in comparison to those treated with mAbE12 (FIGS. 7 d-e). As shown structural changes between E12 treated colonand control are evident. This may be explained by the shift in cytokineprofile from pro-inflammatory (in control treated animals) toanti-inflammatory cytokines (in E12 treated animals) as shown in FIGS. 8a-i.

FIGS. 8 a-c show sections of untreated IBD induced rats. Massivesubmucosal infiltration of macrophages (ED1⁺) and both mucosal andsubmucosal infiltration of T cells (CD3+) are shown. IL-10 productionwas barely detected, mainly in the mucosa.

FIGS. 8 d-f show sections of isotype matching control treated animals.Submucosal infiltration of macrophages (ED1+), mucosal infiltration of Tcells (CD3+) and minor IL-10 production in the mucosa are detected.

FIGS. 8 g-i show sections of E12 treated rats. Submucosal infiltrationof macrophages (ED1+) in damaged areas is shown, and presence ofmacrophages in the lamina propria of unaffected areas is detected. CD3+T cell infiltrate healthy mucosa, with marked IL-10 production at themucosa.

REFERENCES CITED Additional References are Cited in the Text

-   1. Brown M S, Goldstein J L. Lipoprotein metabolism in the    macrophage: implications for cholesterol deposition in    atherosclerosis. Annu Rev Biochem 1983; 52:223-61.-   2. Krieger M. The other side of scavenger receptors: pattern    recognition for host defense. Curr Opin Lipidol 1997; 8:275-80.-   3. Krieger M, Herz J. Structures and functions of multiligand    lipoprotein receptors: macrophage scavenger receptors and LDL    receptor-related protein (LRP). Annu Rev Biochem 1994; 63:601-37.-   4. Platt N, Gordon S. Is the class A macrophage scavenger receptor    (SR-A) multifunctional?—The mouse's tale. J Clin Invest 2001;    108:649-54.-   5. Janeway C A. Approaching the asymptote? Evolution and revolution    in immunology. Cold Spring Harb. Symp. Quant. Biol. 1989; 54:1-13.-   6. Acton S L, Scherer P E, Lodish H F, Krieger M. Expression cloning    of SR-BI, a CD36-related class B scavenger receptor. J Biol Chem    1994; 269:21003-9.-   7. Krieger M. Scavenger receptor class B type I is a multiligand HDL    receptor that influences diverse physiologic systems. J Clin Invest    2001; 108:793-7.-   8. Hyka N, Dayer J M, Modoux C, et al. Apolipoprotein A-I inhibits    the production of interleukin-1β and tumor necrosis factor-α by    blocking contact-mediated activation of monocytes by T lymphocytes.    Blood 2001; 97:2381-9.-   9. Youssef S, Maor G, Wildbaum G, Grabie N, Gour-Lavie A, Karin N.    C—C chemokine-encoding DNA vaccines enhance breakdown of tolerance    to their gene products and treat ongoing adjuvant arthritis. J Clin    Invest 2000; 106:361-71.-   10. Youssef S, Wildbaum G, Maor G, et al. Long-lasting protective    immunity to experimental autoimmune encephalomyelitis following    vaccination with naked DNA encoding C—C chemokines. J Immunol 1998;    161:3870-9.-   11. Wildbaum G, Netzer N, Karin N. Plasmid DNA encoding    IFN-γ-inducible protein 10 redirects antigen-specific T cell    polarization and suppresses experimental autoimmune    encephalomyelitis. J Immunol 2002; 168:5885-92.-   12. Wildbaum G, Westermann J, Maor G, Karin N. A targeted DNA    vaccine encoding fas ligand defines its dual role in the regulation    of experimental autoimmune encephalomyelitis. J Clin Invest 2000;    106:671-9.-   13. Wildbaum G, Youssef S, Karin N. A targeted DNA vaccine augments    the natural immune response to self TNF-α and suppresses ongoing    adjuvant arthritis. J Immunol 2000; 165:5860-6.-   14. Wildbaum G, Karin N. Augmentation of natural immunity to a    pro-inflammatory cytokine (TNF-α) by targeted DNA vaccine confers    long-lasting resistance to experimental autoimmune    encephalomyelitis. Gene Ther 1999; 6:1128-38.-   15. Salomon I, Netzer N, Wildbaum G, Schif-Zuck S, Maor G, Karin N.    Targeting the Function of IFN-γ-Inducible Protein 10 Suppresses    Ongoing Adjuvant Arthritis. J Immunol 2002; 169:2685-93.-   16. Sato Y, Roman M, Tighe H, et al. Immunostimulatory DNA sequences    necessary for effective intradermal gene immunization. Science 1996;    273:352-357.-   17. Raz E, Tighe H, Sato Y, et al. Preferential induction of a Th1    immune response and inhibition of specific IgE antibody formation by    plasmid DNA immunization. Proc Natl Acad Sci USA 1996; 93:5141-5.-   18. Hemmi H, Takeuchi O, Kawai T, et al. A Toll-like receptor    recognizes bacterial DNA. Nature 2000; 408:740-5.-   19. Wen-Ming C, Xing Gong K, Zhi-Wei L, et al. DNA-PKcs Is Required    for Activation of Innate Immunity by Immunostimulatory DNA. Cell    2000; 103:909-918.-   20. Mendel I, Kerlero de Rosbo N, Ben-Nun A. A myelin    oligodendrocyte glycoprotein peptide induces typical chronic    experimental autoimmune encephalomyelitis in H-2b mice: fine    specificity and T cell receptor V β expression of encephalitogenic T    cells. European Journal of Immunology 1995; 25:1951-9.-   21. Yednock T A, Cannon C, Fritz L C, Sanchez-Madrid F, Steinman L,    Karin N. Prevention of experimental autoimmune encephalomyelitis by    antibodies against α4β1 integrin. Nature 1992; 356:63-6.-   22. Wildbaum G, Netzer N, Karin N. Tr1 cell-dependent active    tolerance blunts the pathogenic effects of determinant spreading. J    Clin Invest 2002; 110:701-10.-   23. Husemann J, Silverstein S C. Expression of scavenger receptor    class B, type I, by astrocytes and vascular smooth muscle cells in    normal adult mouse and human brain and in Alzheimer's disease brain.    Am J Pathol 2001; 158:825-32.-   24. McRae B L, Kennedy M K, Tan L J, Dal Canto M C, Picha K S,    Miller S D. Induction of active and adoptive relapsing experimental    autoimmune encephalomyelitis (EAE) using an encephalitogenic epitope    of proteolipid protein. Journal of Neuroimmunology 1992; 38:229-40.-   25. Karin N, Binah O, Grabie N, et al. Short peptide-based    tolerogens without self-antigenic or pathogenic activity reverse    autoimmune disease. J Immunol 1998; 160:5188-94.

What is claimed is:
 1. A method of treating an inflammatory disease in asubject in need thereof, the method comprising administering to saidsubject a therapeutically effective amount of an isolated polypeptidecomprising an antigen recognition domain capable of specifically bindinga human scavenger receptor, wherein said antigen recognition domaincomprises CDR amino acid sequences as set forth in SEQ ID NO: NO: 11,15, 19, 23, 27 and 31, thereby treating the inflammatory disease in thesubject.
 2. The method of claim 1, wherein said inflammatory disease isan autoimmune inflammatory disease.
 3. The method of claim 2, whereinsaid autoimmune inflammatory disease is selected from the groupconsisting of Systemic Lupus Erythematosus (SLE), Amyotrophic LateralSclerosis (ALS) and Rheumatoid Arthritis (RA).
 4. The method of claim 2,wherein said autoimmune inflammatory disease is Systemic LupusErythematosus (SLE).
 5. The method of claim 2, wherein said autoimmuneinflammatory disease is Amyotrophic Lateral Sclerosis (ALS).
 6. Themethod of claim 2, wherein said autoimmune inflammatory disease isRheumatoid Arthritis (RA).